Widefield fluorescence
The standard widefield arrangement: the whole field is lit through the objective and imaged onto a camera through a filter cube. Fast and light-efficient, but it collects out-of-focus light from the full sample depth.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Epifluorescence
The standard widefield arrangement: the whole field is lit through the objective and imaged onto a camera through a filter cube. Fast and light-efficient, but it collects out-of-focus light from the full sample depth.
How it differs from the standard method
- LED or lamp illumination through a filter cube.
- Resolution ≈ 0.51 λ/NA laterally; no optical sectioning.
- Choose magnification and binning so the pixels meet Nyquist.
1Light in, different light out
A fluorophore absorbs a photon of one colour and, a few nanoseconds later, emits one of a longer wavelength. A widefield fluorescence microscope lights the whole field at once through the objective (epi-illumination) and uses a filter cube to separate the two colours: an excitation filter picks the band that excites the dye, a dichroic mirror reflects it down onto the sample and passes the longer-wavelength emission back up, and an emission filter cleans that up before the camera.
Set by NA
The image of a point is a spot about 0.51 λ/NA wide (FWHM), roughly 190 nm for GFP with a 1.4 NA oil objective and twice that with a 0.75 NA dry lens.
Pixels at Nyquist
The camera pixel in the sample is 6.5 µm divided by the magnification (times any binning). It needs to be about 2.3 times smaller than the resolution, or detail is lost to the pixels.
NA⁴ / M²
Epi-illumination brightness rises steeply with NA and falls with magnification: a high-NA objective is both sharper and brighter.
Shot noise
Signal-to-noise is set by how many photons each pixel collects: about √N for N photons, plus the camera's read noise.
2The light budget
Every photon you collect costs the sample: fluorophores bleach after a certain number of excitation cycles, and intense blue light damages living cells. Power × exposure sets both the signal and the damage.
3What widefield cannot do
The whole thickness of the sample is lit, and light from above and below the focal plane arrives at the camera as a blur. In thin, flat cells this is a soft glow; in tissue or rounded cells it hides the in-focus detail.
4TIRF
When light in glass (n 1.52) hits the boundary with the cell (n about 1.37) at more than the critical angle, about 64.5°, it is totally reflected. A thin evanescent field leaks across the boundary and decays exponentially, with a 1/e depth of about 70–200 nm depending on angle and wavelength.
Key terms
Practise these as flashcards →Excitation filter, dichroic mirror and emission filter matched to one fluorophore.
n × sin of the objective's half-angle; it sets resolution (∝ λ/NA) and light collection.
Pixels at least 2–3 times smaller than the resolution, so the image preserves what the optics deliver.
Adding neighbouring pixels together on the camera: more signal per pixel, coarser sampling.
The most electrons a pixel can hold; brighter pixels clip and lose their true intensity.
Irreversible loss of fluorescence with light dose; it makes later frames dimmer than earlier ones.
The incidence angle above which light is totally internally reflected, asin(n₂/n₁), about 64.5° from glass into cytoplasm.
The exponentially decaying light beyond a totally reflecting surface that excites only the first ~100 nm in TIRF.
When to use it
- Imaging fluorescent proteins or dyes in thin cultured cells, live or fixed, quickly and with modest light dose.
- Time-lapse of fast events across a whole field, where a camera beats a scanning confocal for speed and photon efficiency.
- TIRF for events at the plasma membrane facing the glass: endocytosis, exocytosis, adhesion, single-molecule tracking.
Compared with alternatives
Rejects out-of-focus light with a pinhole; choose it for thick samples and 3D stacks.
Breaks the ~200 nm limit when the structure is smaller than widefield can resolve.
Adds the environmental control and scheduling needed to follow cells for hours.
How fixed samples are labelled before they are imaged on a widefield microscope.
Choosing the label: brightness, photostability and spectrum decide much of the light budget.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
The standard widefield arrangement: the whole field is lit through the objective and imaged onto a camera through a filter cube. Fast and light-efficient, but it collects out-of-focus light from the full sample depth.
- LED or lamp illumination through a filter cube.
- Resolution ≈ 0.51 λ/NA laterally; no optical sectioning.
- Choose magnification and binning so the pixels meet Nyquist.